Amplification of spatially isolated adenosine pathway by tumor–macrophage interaction induces anti-PD1 resistance in hepatocellular carcinoma

Background Immune checkpoint blockade resistance narrows the efficacy of cancer immunotherapies, but the underlying mechanism remains elusive. Delineating the inherent mechanisms of anti-PD1 resistance is important to improve outcome of patients with advanced HCC. Method The level of cricTMEM181 was measured in HCC patients with anti-PD1 therapy by RNA sequencing and then confirmed by qPCR and Sanger sequencing. Immune status in tumor microenvironment of HCC patients or mice models was evaluated by flow cytometry and IHC. Exosomes from HCC cell lines were isolated by ultracentrifugation, and their internalization by macrophage was confirmed by immunofluorescence. The underlying mechanism of HCC-derived exosomal circTMEM181 to macrophage was confirmed by SILAC, RNA FISH and RNA immunoprecipitation. The ATP–ADO pathway amplified by HCC–macrophage interaction was evaluated through ATP, AMP and ADO measurement and macrophage-specific CD39 knockout mice. The role of circTMEM181 in anti-PD1 therapy and its clinical significance were also determined in our retrospective HCC cohorts. Results Here, we found that circTMEM181 was elevated in hepatocellular carcinoma (HCC) patients responding poorly to anti-PD1 therapy and in HCC patients with a poor prognosis after operation. Moreover, we also found that high exosomal circTMEM181 favored the immunosuppressive microenvironment and endowed anti-PD1 resistance in HCC. Mechanistically, exosomal circTMEM181 sponged miR-488-3p and upregulated CD39 expression in macrophages. Using macrophage-specific CD39 knockout mice and pharmacologic approaches, we revealed a novel mode of anti-PD1 resistance in HCC. We discovered that cell-specific CD39 expression in macrophages and CD73 expression in HCC cells synergistically activated the eATP–adenosine pathway and produced more adenosine, thereby impairing CD8+ T cell function and driving anti-PD1 resistance. Conclusion In summary, HCC-derived exosomal circTMEM181 contributes to immunosuppression and anti-PD1 resistance by elevating CD39 expression, and inhibiting the ATP–adenosine pathway by targeting CD39 on macrophages can rescue anti-PD1 therapy resistance in HCC. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13045-021-01207-x.

to the protocols. Targeted miRNAs were normalized to U6B RNA levels using the 2 -ΔCt model.
Targeted mRNA or circRNA levels were adjusted using GAPDH. qPCR assays were performed in triplicate for each sample, and the mean value ± SD was used for the statistics of RNA expression levels.

Western blot
Cells or tissues were lysed by RIPA lysis buffer (Yeasen Biotechnology (Shanghai), Co. Ltd.) and the proteins were quantified using the BCA Kit. Proteins were separated on 10 or 12.5 % SDS-PAGE gels, transferred to PVDF membranes (Millipore) and blocked with Protein Free Rapid Blocking Buffer (Epizyme Biotech). After incubating overnight at 4 °C with the primary antibodies described in the Key Resources Table, the PVDF membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Immersed the PVDF membranes with ECL Detection Reagent (Yeasen Biotechnology). The immunoblots were then detected using a gel image analysis system.

RNA ISH and RNA FISH
Formalin-fixed paraffin-embedded slides were firstly incubated at 70°C for 40 min, then performed deparaffinization following dimethylbenzene and graded alcohol, rinsed with cold tap water for three times. The slides were then boiled in the retrieval solution for 15 minutes. After cooling to room temperature, added proteinase K(20 μg/ml) to cover the tissue and incubated at 37℃ for 8 minutes. Washed three times with PBS. Added Pre-hybridization solution to each section and incubated for 1 h at 37℃. Discarded the pre-hybridization solution, and the slides were hybridized with probes for the targeted nucleic acid overnight at 40°C. Washed the slides with 5 ×, 1 ×, and 0.2 × saline-sodium citrate (SSC) at room temperature.
For FISH, stained cell nuclei by incubating with DAPI for 5 min in the dark, and then mounting with anti-fluorescence quenching sealing tablets. Microscopic examination and photography were then performed.
For ISH, added blocking solution to the section and incubated at room temperature for 30 min. After removing the blocking solution, anti-DIG-HRP was added and incubated at 37 ℃ for 40 min. Then, washed the sections in TBS four times for 5 min each. Dried sections slightly, and added NBT/BCIP to mark the tissues, finally stopped the reaction by wash in running tap water.

Cell lines and cell culture
All the identified cell lines were provided from Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education of P.R.C or National Collection of Authenticated Cell Cultures, P.R.C. Cell lines HepG2, HCCLM3, MHC97H, Huh-7, PLC/PRF/5, H22, Hepa1-6 were cultured in DMEM with 10% fetal bovine serum. The cell lines THP-1 and Li-7 were cultured in RPMI-1640 with 10% fetal bovine serum. All the cells were cultured at 37 °C in a 5% CO2 incubator.

Clonogenic assay
Clonogenic assay were performed in six-well plates and seeded cells for each plate. After culturing for 2 weeks, removed the medium above the cells and rinsed carefully with PBS for three times.
Then removed the PBS and add 2 ml 4% paraformaldehyde. After waiting for 15 min, removed the 4% paraformaldehyde and rinsed with PBS for three times. Added Crystal Violet Staining Solution (0.5%) and leaved this for 20 min. Removed the Crystal Violet Staining Solution carefully and rinse with PBS. Leaved the plates to dry in normal air at room temperature for 1 day. Counted the number of colonies under the microscope.

Transwell assays and cell co-culture
For Transwell assay, medium with 10% fetal bovine serum were added to the multiwell plate, followed by adding the Transwell inserts, and lastly adding the cells with medium without fetal bovine serum to the inside compartment. After 3-7 days, the Transwell inserts were removed and rinsed with PBS for three times. Then the Transwell inserts were immersed in 4% paraformaldehyde for 15 min and the cells inside compartment were wiped with a cotton swab. Rinsed with PBS for three times, added Crystal Violet Staining Solution (0.5%) and leaved this for 20 min. Washed the Crystal Violet and counted the number of the cells under the microscope after PBS wash.
For cell co-culture, one of the cells was added to the bottom of the multiwell plate with 10% fetal bovine serum and other needed reagents, followed by adding the Transwell inserts (pore size=0.4 μm), and finally added the second cell with the medium to the inside compartment.

Immunohistochemistry and multiplex immunofluorescence
Formalin-fixed paraffin-embedded slides or TMA slides were incubated at 70°C for 40 min, then performed deparaffinization following dimethylbenzene and graded alcohol (95%, 85%,75%), rinsed with PBS for three times. Antigen retrieval was then performed by EDTA Antigen Retrieval Solution or Citrate Antigen Retrieval Solution at 95°C for 15 min. Rinsed with PBS for three times and blocking endogenous enzymes by Endogenous Peroxidase Blocking Buffer for 10-25 min.
Rinsed with PBS for three times and incubated with Normal Goat Serum For Blocking for 20 min.
Removed the blocking solution on the slide, and added the primary antibody working solution to completely cover the sample. Incubated at 4℃ overnight. Washed the slides for 3 times with 1× TBST for 2 min each time. After removing the washing solution remaining on the slide, added HRP secondary antibody working solution, and immersed the sample area. Incubated at room temperature for 10 minutes and washed the slides 3 times with 1 × TBST.
Removed the washing solution remaining on the glass slide and added 1 × NEON TSA fluorescent dye to immerse the sample area. Incubated at room temperature for 5 min and washed the slides 3 times with 1 × TBST.
For multiplex immunofluorescence, repeated the steps above since antigen retrieval with different primary antibodies and fluorescent dye. Added anti-fluorescence quenching sealing tablets with DAPI and then mounted the slide with a cover glass. Imaging was performed by Pannoramic MIDI (3DHISTECH Ltd.).
For Immunohistochemistry, after washing the HRP secondary antibody, dried sections slightly, and added DAB to mark the tissue for 10 min. Added Hematoxylin staining solution, staying for 50 sec and washed in tap water. Immersed the slide in water for 30 min. Performed dehydrate following gradient ethanol (75%, 85%, 100%) and dimethylbenzene. Finally, mounted the slide with resin mounting medium and covered glass.

Mice model and in vivo tumor models
Male C57BL/6J mice aged 6-week-old were purchased from Vitalriver Laboratory Animal Co., Ltd The tumors were screened by optical in vivo Imaging system. Treatment was performed 7 days later after tumor construction. Anti-mouse PD1 was intraperitoneally injected (10 mg/kg, 3 times every week, for 2 weeks). POM1 was intraperitoneally injected (5 mg/kg, 4 times every week, for 2 weeks). Clophosome was intraperitoneally injected (0.2 μl/mouse, every week, for 2 weeks).

ATP, AMP, ADP and Adenosine measurement
Prepared standard products (ATP, AMP, ADP and adenosine) and dissolved in concentration of 10 μg/mL with ultrapure water. Injected 10 μl to the machine (Agilent 1260 Infinity II) and record the chromatogram. Added 300 μl Extract 1 to 200 μl sample and stayed at 4℃ for 1 h. Centrifuged at 4℃, 15, 000 rmp for 10 min and took 200 μl of the supernatant, add an equal volume of Extract 2, mix well, centrifuge at 15,000 rmp for 10 min, take the supernatant, and filter with a 0.22um needle filter. Inject 10ul on the machine and record the chromatogram. The chromatogram acquisition and integration of the compound is performed by the software Chemstation (Agilent).

ELISA
Supernatants were collected from medium, and IL-10 and TNF-α were analyzed using ELISA kits (R&D Systems) according to the instructions.

RNA sequencing
RNA sequencing and bioinformatics analysis were performed by Majorbio (Shanghai, China). In brief, tumor tissue or cell samples were subjected to total RNA extraction with TRIzol reagent. For circRNA sequencing, ribosomal RNAs and linear RNAs were removed by Ribo-Zero Gold rRNA Removal Kit and RNase R to enrich circRNA. Enriched RNAs were amplified and transcribed to create the final cDNA library. Next generation sequencing cDNA libraries was performed on the Illumina PE150 platform.

SILAC (stable isotope labeling by amino acids in cell culture)
SILAC (stable isotope labeling by amino acids in cell culture) was performed according to the protocol (doi: 10.1038/nprot.2006.427). In brief, to investigate different cell protein profiles, THP-1 co-exoOE was cultured in a growth medium with 'heavy' amino acids and THP-1 co-exoCtl was cultured in a growth medium with 'light' amino acids. While a reverse SILAC experiment that switched heavy and light medium was also performed. The cells were collected and mixed ('heavy': 'light'=1:1), and analyzed with mass spectrometry. Intensity of MS signals between 'light' and 'heavy' peptides was identified to show the different protein abundance between cells.

Optical in vivo Imaging
Mice were anaesthetized with sodium pentobarbital. Then the mice were administrated D-Luciferin (dissolved in PBS, 150 mg/kg) by intraperitoneal injection. After waiting for 15 min, fluorescence imaging was performed by the imaging system IVIS Lumina K (PerkinElmer) with the software Living Image 4.4.

Exosome isolation
Balanced the sample of medium, cell lysis or serum with PBS, and centrifuged at 1, 500 g, 4°C for 15 minutes. The supernatant then centrifuged at 10, 000 g, 4°C for 30 minutes. Acquired the supernatant after centrifugation and centrifuged for 30 minutes at 14, 000 g, 4°C. After centrifugation, transferred the supernatant to an ultra-high-speed centrifuge tube, and used a pipette to balance with 1×PBS buffer on a balance that can be accurate to 0.001 g, and carefully placed it in the ultra-high-speed centrifuge at 4°C, 110, 000 g ultra-high-speed centrifugation for 120 minutes.
After centrifugation, carefully discarded the supernatant, resuspended the pellet in 1 × PBS buffer, and centrifuged at 4°C, 110,000 g, ultra-high speed for 60 minutes. After centrifugation, the supernatant was carefully discarded. The remaining liquid at the bottom of the centrifuge tube was the exosomes. Used 300 μl of 1 × PBS to carefully blow the bottom of the centrifuge tube and suck it into the 0.5-1.5 ml centrifuge tube.

Dual luciferase reporter assay
HEK 293T cells were co-transfected with a luciferase reporter vector and miR-488-3p or miR-1298 mimics or the negative control using Lipo-fectamine 2000 reagent. After 48 h, Firefly and Renilla luciferase signals were then determined by dual-luciferase reporter assay (Promega, USA).

Tumor dissociation
Tumor dissociation were performed following the protocol from Tumor Dissociation Kit, mouse Miltenyi Biotec) or Tumor Dissociation Kit, human (#130-095-929, Miltenyi Biotec). In brief, enzymes were prepared by reconstitution of the lyophilized powder, then we prepared enzyme mix by adding the components into a gentleMACS™ C Tube, including DMEM and the enzymes. Tumor tissue from mice or HCC patients was cut into small pieces and transferred into the C Tubes. Then the gentleMACS™ Dissociator were used to perform dissociation program.
After termination of the program, the C Tubes containing tumor tissue were detached and incubated at 37 °C under continuous rotation. After incubation for certain time according to the protocol, resuspended sample and applied the cell suspension to a strainer (70 µm) placed on a 50 ml tube.
Washed the strainer with 10 ml RPMI 1640 and centrifuged cell suspension at 35 0 × g for 5 min.
Aspirated supernatant and performed further experiment.

Flow cytometry and sorting
Isolated single-cell suspensions from the blood or tissue of human or mouse were centrifuged (350g, 5min, at 4°C) in a centrifuge and resuspended in staining buffer (1% FBS in PBS) on ice. Calculated the cells and transferred 10 6 cells into each staining tube. All the samples were placed on ice throughout the staining procedure and single-cell suspensions were washed with PBS (4°C) for two times and resuspended in 20 μl staining buffer (1% FBS in PBS). FcBlocks (1μl for each tube) were used to incubate with the single-cell suspensions for 15 min before staining.
For surface staining, the antibodies were incubated with cell suspensions for 20 min in dark place at 4°C. For intracellular staining, saponin was used after fixation by 4% paraformaldehyde, and the antibodies were incubated with cell suspensions for 20 min in dark place at 4°C. For nuclear staining, after fixation and permeabilization by buffer set, the antibodies were incubated with cell suspensions for 20 min in dark place at 4°C.
For staining of peripheral blood samples, after staining, red blood cells were lysed for 4 min at room temperature in red blood cell lysing buffer. Cell suspensions were washed with PBS (4°C) for two times and filtered using a strainer (70 µm) before flow cytometry analysis. Data was analyzed by FlowJo 10.6.2 software.
The surface staining for sorting was the same as that for flow cytometry analysis, except that all the procedure was operated in a sterile environment.